1. Field of the Invention
This invention relates to identifying particular Trichoderma asperellum isolates as biocontrol agents and their use for reducing Phytophthora ramorum growth in soil, for the remediation of P. ramorum-infested soil in commercial nurseries, and for the protection of potted, rooted plants from infestation and spread of P. ramorum. The T. asperellum isolates control soilborne plant disease by reducing P. ramorum populations and suppressing the spread and reemergence of P. ramorum in the soil. The invention relates to particular T. asperellum isolates screened for their mycoparasitic characteristics and effective antagonistic activity for P. ramorum. The present invention relates to a biocontrol method comprising the application of a particular T. asperellum isolate to soil infested with P. ramorum, such as commercial nursery soil, to obtain remediated soil, i.e., soil where P. ramorum is not detectable. Potting soil amended with T. asperellum isolate 04-22 protects uninfected plants from P. ramorum infestation and suppresses sporulation and further infection of P. ramorum infested plants.
2. Description of the Relevant Art
Phytophthora ramorum Werres, de Cock, and Man in't Veld, causal agent of sudden oak death (SOD) and ramorum blight, is a pathogen of worldwide concern. Phytophthora ramorum is known to have broad host range based upon isolations from natural and artificial laboratory infections (USDA/APHIS. 2012. Retrieved from the Internet Mar. 18, 2013: <URL: aphis.usda.gov/plant_health/plant_pest_info/pram/downloads/pdf_files/usdaprlist.pdf). In the United States, where it is a regulated quarantine organism, P. ramorum has a limited distribution in forests in coastal California and Oregon. P. ramorum was first confirmed in a US nursery in 2001 in Santa Cruz County, Calif. (Frankel, S. J. 2008. Austral. Plant Path. 37:19-25). Despite regulations on the movement of P. ramorum host material from known infested Californian counties, the pathogen was detected in 20 different states by the end of 2004 (Cooperative Agriculture Pest Survey Program. 2005. Phytophthora ramorum 2004 National Nursery Survey. USDA-APHIS, Plant Protection and Quarantine. 15 pp.). To date, on the east coast, P. ramorum has only been isolated from nursery sites (and in a few instances, from residential sites where infected nursery stock had been planted) and from adjacent bodies of water (Cave et al. 2008. Retrieved from the Internet Mar. 18, 2013: <URL: aphis.usda.gov/plant_health/plant_pest_info/pra-cphst-08.pdf; Jeffers et al. 2010. In: Proceedings of the Sudden Oak Death Third Science Symposium, eds. Frankel et al., General Technical Report PSW-GTR-229, Albany, Calif.: Pacific Southwest Research Station, Forest Service, USDA, pp. 69-71). However, there are many susceptible forest hosts, leading to a high potential for spread to forested areas.
Ramorum blight, the disease of ornamental plants caused by P. ramorum, is considered to have only a moderate impact on ornamental plants since it is usually not lethal to these hosts (Kliejunas, J. T. 2011. In: A Risk Assessment of Climate Change and the Impact of Forest Diseases on Forest Ecosystems in the Western United States and Canada. General Technical Report PSW-GTR-235, Albany, Calif.: Pacific Southwest Research Station, Forest Service, USDA). Symptoms on rhododendrons, camellias, viburnums, and other hosts are usually non-distinctive leaf necrosis and blights that are not easily distinguishable from other diseases. However, more importantly, ramorum blight has a serious impact on the nursery industry. The continued detections of P. ramorum on ornamental nursery stock (Tubajika et al. 2006. Plant Health Progress doi: 10.1094/PHP-2006-0315-02-RS) has increased concern about the potential spread of this pathogen throughout the nursery industry. Because long distance dispersal of P. ramorum is facilitated by shipments of infected nursery plants across the country (Parke and Lucas. 2008. Retrieved from the Internet Sep. 6, 2012: <URL: The Plant Health Instructor. Doi: 10.1094/PHI-I-2008-0227-01) and because concerns exist that the pathogen could spread from nurseries into natural habitats (Davidson et al. 2005. Phytopath. 95:587-596), strict guidelines have been imposed on nurseries in areas known to have P. ramorum. These restrictions have had substantial economic consequences for the nursery industry (Kliejunas, supra).
Once P. ramorum is detected in a nursery, a set of procedures (the APHIS Confirmed Nursery Protocol) must be carried out to eradicate the pathogen (USDA/APHIS. 2005. Retrieved from the Internet: <URL: aphis.usda.gov/plant_health/plant_pest_info/pram/downloads/pdf_files/CNPv7.pdf). However, remediation of infested nurseries has proved problematic. There have been recurrent infections of plants in nurseries where diseased plants were detected and removed. In 2005, the California Department of Food and Agriculture identified 53 P. ramorum-positive California nurseries, eight of which had recurrent infections; in 2006, 24 recurrent nurseries were reported; in 2007, five were reported (California Oak Mortality Task Force [COMFT], 2009. Retrieved from the Internet April, 2009: <URL: nature.berkeley.edu/comtf/html/chronology.htm). It was not always clear if the recurrence was due to a new introduction of diseased stock, or to infection of healthy plants from some source of inoculum persisting in the nursery.
Studies have shown that P. ramorum has a soilborne phase and can persist in soil and potting mix, thus playing an important role in survival and infection of new hosts (Shishkoff, N. 2007. Plant Dis. 91:1245-1249; Linderman and Davis. 2006. HortTech. 16:502-507). Resistant spores, such as P. ramorum chlamydospores, can survive in soil for long periods of time (Erwin and Ribeiro. 1996. Phytophthora Diseases Worldwide. APS Press, St. Paul, Minn.). The pathogen has been recovered from the soil directly below containerized plants (Jeffers, S. 2005. Phytopath. 95: S48; Tjosvold at al. 2009. Plant Disease 93:371-376; Dart et al. 2007. Plant Dis. 91:1419-1422), from litter on the ground (Neubauer at al. 2006. Gesunde Pflanzen 58:185-191), as well as from the rootball of an asymptomatic non-host plant (Dart and Chastagner. 2007. Retrieved from the Internet: Plant Health Progress doi:10.1094/PHP-2007-0816-01-BR). Further, P. ramorum was found not to be limited to the organic layer of soil, as it has been detected in 10-cm deep soil samples (Dart at al., supra). The continuing spread and reemergence of P. ramorum from infested nursery stock has emphasized the difficulty in eliminating this pathogen completely with current management practices such as treating with metam-sodium, attempting eradication, removing large quantities of soil, or shutting down the nursery completely (Frankel, supra; Yakabe and MacDonald. 2008. In: Proceedings of the Sudden Oak Death Third Science Symposium, eds. Frankel et al., General Technical Report PSW-GTR-214, Albany, Calif.: Pacific Southwest Research Station, Forest Service, USDA, pp. 113-114).
In order to remove restrictions on individual nurseries, these establishments must prove that they no longer have P. ramorum by testing the soil and plant material (USDA/APHIS. 2010. Retrieved from the Internet Sep. 6, 2012: <URL: aphis.usda.gov/plant_health/plant_pest_info/pram/downloads/CNP/CNP %20v8.0%201-2011.pdf). If P. ramorum-infected plant material is found, the required protocol dictates that all host and associated host plants within a defined destruction block be destroyed (USDA/APHIS 2010, supra). In addition, a 10-meter radius around the destruction block is designated as a quarantine block, where plants cannot be moved in or out, and must be maintained in that status for a minimum period of 90 days to determine if P. ramorum has spread beyond the border of the destruction block. Ornamental plants often are cultivated under high density conditions that could easily facilitate plant-to-plant spread of P. ramorum outside the initial destruction block (Englander and Tooley. 2003. Retrieved from the Internet Sep. 6, 2012: <URL:apsnet.org/online/SOD.doi: 10.1094/SOD-2003-LE). P. ramorum has been demonstrated to spread plant-to-plant in aerial infections by physical contact and splash from rain and overhead irrigation with symptoms often taking some time to be noticed (Heungens et al. 2010. In: Proceedings of the Sudden Oak Death Fourth Science Symposium, eds. Frankel et al., General Technical Report PSW-229, Albany, Calif.: Pacific Southwest Research Station, Forest Service, USDA, pp. 72-75; Tjosvold et al. 2008. Retrieved from the Internet Sep. 6, 2012: <URL: Plant Health Progress. Doi:10.1094/PHP-2008-01-RS).
Different techniques, such as pressure washing or chemical sanitization, are used by growers to attempt to decontaminate used containers. Aerated steam and chemical fumigants are known methods to eliminate soilborne pathogens. Linderman and Davis (2008a. HortTech. 18:106-110) found that P. ramorum populations in potting media were killed by aerated steam heat treatments of 50° C. or higher or treatment with metam sodium concentrations of 0.25 ml per liter of medium. However, using these techniques increases the chance of destroying beneficial microorganisms and of working in a hazardous environment.
It is essential to controlling the spread of this pathogen that infested nurseries be successfully cleaned of all propagules of the pathogen. Although remediation studies using chemical sterilants have been done in situ in infested nurseries in California (Yakabe and MacDonald, supra), there was no opportunity at that time to set up control plots where soil was left untreated; therefore, it was difficult to evaluate efficacy of the treatments. In addition, chemical sterilants, while often effective, have limitations because of their toxicity; many cannot be used in residential areas. Therefore, safer alternatives would be a necessary addition to the list of possible control measures in a good integrated pest management system.
There have been numerous studies examining the use of fungicides to control P. ramorum on ornamentals (Heungens et al. 2006. In: Proceedings of the Sudden Oak Death Second Science Symposium, eds. Frankel et al., General Technical Report PSW-196, Albany, Calif.: Pacific Southwest Research Station, Forest Service, USDA, pp. 241-257; Linderman and Davis. 2008b. Plant Health Progress. Doi:10.1094/PHP-2008-2011-01-RS; Tjosvold at al. 2008, supra; Pérez-Sierra et al. 2011. Plant Path. 60:1069-1076). Although fungicides are effective to some degree when applied as a preventative (Heungens at al. 2006, supra; Tjosvold et al. 2008, supra), they require repeated applications. Linderman and Davis (2008b, supra) found that all chemicals tested in their study were fungistatic and not fungicidal. Besides environmental concerns, repeated applications potentially could lead to fungicide resistance by the pathogen as has been observed by other Phytophthora spp. (Grünwald at al. 2006. Phytopath. 96:1397-1403). In addition, U.S. and European Union regulations prohibit the application of fungicides in the quarantine zones (USDA/APHIS 2010, supra; Pérez-Sierra et al., supra) due to potential masking of symptoms. These concerns have lead to the desire to investigate other control options, including biological control.
Biological control through the use of microorganisms is a method to manage plant diseases that has gained a lot of interest due to its sustainability and low negative impacts on the environment. In natural soil, oomycetes, chytrids, actinomycetes, hyphomycetes, and bacteria were observed to parasitize oospores of different Phytophthora spp. (Sneh at al. 1977. Phytopath. 67:622-628). Some of the most studied and promising fungi used in a biocontrol system are Trichoderma spp. (Harman at 2004. Nature Rev. Microbiol. 2:43-56; Smith at al. 1990. Phytopath. 70:880-885).
Populations of Trichoderma spp., which are often abundant in composts and compost-amended media, typically suppress Pythium and Phytophthora root rots within days after their formulation (De Ceuster and Hoitink. 1999. Compost Sci. Util. 7:6-15.; Hoitink and Boehm. 1999. Ann. Rev. Phytopath. 37:427-446). Trichoderma spp. are reported to suppress soilborne diseases caused by Rhizoctonia solani Kuhn and Phytophthora spp. in containerized systems (Chung and Hoitink. 1990. Phytopath. 80:73-77; da S. Costa at al. 2000. Brazilian J. Microbiol. 31:239-246; Sharifi Tehrani and Nazari. 2004. Acta Horticulturae 635:137-139). Phytophthora capsici populations were reduced when infested soil was amended with T. harzianum (Ezziyyani at al. 2007. J. Phytopath. 155:342-349; Sid Ahmed et al. 1999. Plant Path. 48:58-65). Mycoparasitism by the Trichoderma spp. was shown to be the primary mode of action that reduced populations of various pathogenic soil fungi (Gupta et al. 1999. J. Phytopath. 147:19-24; Watanabe et al. 2007. J. Pesticide Sci. 32:222-228). Currently, T. asperellum is being studied as a biological control agent to manage black pod disease of cacao in Cameroon. Recent results show that disease incidence was lower when T. asperellum was applied on infected cacao trees (Tondje et al. 2007. Biol. Control. 43:202-212).
While these various biocontrol methods and formulations for effective control of plant diseases, including those diseases caused by Phytophthora, are known in the art, there still remains a need for effective biocontrol agents for controlling P. ramorum populations and for suppressing the spread of P. ramorum-contaminated soil to nurseries and non-commercial areas such as residential and forested soil so that drastic measures such as repeated destruction of plants, removal of large quantities of soil or complete closure of nursery locations are not employed. The present invention, described below, provides a particular, effective Tricoderma asperellum isolate and methods of using this isolate to effectively remediate P. ramorum-infested soil and to protect potted, rooted plants from infection and spread of P. ramorum. 